2-Methoxyestradiol Induces Cell Cycle Arrest and Mitotic
Cell Apoptosis in Human Vascular Smooth Muscle Cells
Yu Gui, Xi-Long Zheng
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Abstract—It has been shown that 2-methoxyestradiol (2-ME) inhibits cell proliferation and DNA synthesis in human aortic
smooth muscle cells. However, the cellular mechanisms underlying the antiproliferative activity of 2-ME are unclear.
The present study was performed to explore the cellular mechanisms whereby 2-ME leads to growth inhibition and
apoptosis of human smooth muscle cells. Our results demonstrate that at 1 hour of treatment, 1 ␮mol/L 2-ME induces
multiple spindles, overamplified centrosomes, and multipolar cytokinesis, whereas 10 ␮mol/L 2-ME causes completely
damaged spindle, disorientated centrosomes, and missegregated chromosomes. At 6 hours of treatment, the mitotic
index was increased and reached a maximal level, and cells with 4N DNA content (4N cells) began to accumulate. The
increased mitotic cells induced by 2-ME were apoptotic as detected by both annexin V and TUNEL staining. Blockage
of cells in G1/0 phase by thymidine prevented 2-ME–induced apoptosis. In addition, the increased mitotic index declined
concurrently when even more 4N cells accumulated at 12 to 48 hours of treatment with 10 ␮mol/L 2-ME. Furthermore,
in response to 2-ME, cells delayed entry into the next cell cycle and exhibited aneuploidy or micronuclei. Some
aneuploidy cells continued to synthesize DNA. We conclude that 2-ME treatment not only arrests cells in mitosis and
promotes mitotic cell apoptosis, but also causes cells to undergo “mitotic slippage” and endoreduplication. The
induction of mitotic cell arrest and apoptosis may be a major cellular mechanism by which 2-ME inhibits proliferation
of human smooth muscle cells. (Hypertension. 2006;47:1-10.)
Key Words: apoptosis 䡲 estrogen 䡲 muscle, smooth vascular
S
mooth muscle cell (SMC) proliferation and apoptosis
play important roles in the pathogenesis of vascular
proliferative diseases, such as atherosclerosis and restenosis.
Recent human and animal experimental studies indicate that
cell cycle inhibition holds great potential as a therapeutic
strategy for vascular proliferative diseases.1 Regulation of the
cell cycle is considered a key mechanism for controlling
SMC proliferation.
Cell cycle progression into G1, S, G2, and M phases is
controlled by cell cycle checkpoints that ensure the correct
order and timing of transitions.2 During mitosis, the spindle
assembly checkpoint monitors segregation of sister chromatids, inhibiting the onset of anaphase until all of the chromosomes are properly attached to the mitotic spindle apparatus.3
It has been shown that treatment with microtubule-interfering
agents, such as nocodazole and taxol, induces preanaphase
arrest in early mitosis resulting from effects on the mitotic
spindle.4 This can be followed by tetraploidy arrest because
of aberrant exit from mitosis without sister chromatid segregation and cytokinesis, a process known as “mitotic slippage.”5 As a result of mitotic failure, cells undergo apoptosis.
In addition, after mitotic slippage induced by prolonged
exposure to microtubule-interfering agents, cells may inappropriately continue the next cell cycle and enter S phase with
4N DNA content, a process known as endoreduplication.6
Endoreduplication can be prevented by inhibition of cyclin
E/cyclin-dependent kinase (CDK) 2 activity through induction of p21, a CDK inhibitor.7
2-methoxyestradiol (2-ME), a metabolite of the endogenous estradiol, is present in human blood at concentrations
ranging from picomolar to tens of nanomolars.8 2-ME exhibits antiproliferative and antiangiogenic activity in a variety of
cells and an ex vivo model,9 suggesting that 2-ME could be
a novel antiangiogenic and antiproliferative therapeutic agent.
Additionally, it appears that the antiproliferative activity of
2-ME results mainly from triggering of apoptosis. Recently,
2-ME was found to mediate estradiol-induced inhibition of
DNA synthesis and cell proliferation in human aortic SMCs
in an estrogen receptor–independent manner.10 However, the
mechanism underlying the antiproliferative activity of 2-ME
is not clearly understood. Given that 2-ME can interact with
tubulin directly at the colchicine-binding site and inhibit
tubulin polymerization,11 we hypothesized that the antiproliferative action of 2-ME in human SMCs may result from
mitotic cell arrest and apoptosis through disruption of the
mitotic spindle apparatus.
Our present studies were designed to investigate the
cellular mechanisms by which 2-ME inhibits proliferation of
Received October 12, 2005; first decision October 27, 2005; revision accepted November 29, 2005.
From the Smooth Muscle Research Group, Department of Biochemistry and Molecular Biology, The University of Calgary, Alberta, Canada.
Correspondence to Xi-Long Zheng, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital
Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail [email protected]
© 2005 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.116110.1161/01.HYP.0000199656.99448.dc
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human vascular SMCs. In response to treatment with 2-ME,
cell cycle profiles were first examined by laser scanning
cytometry (LSC). Mitotic spindles, centrosomes, and chromosomes were inspected by immunofluorescent staining and
confocal microscopy. Apoptosis was evaluated by annexin V
apoptotic assay and TUNEL assay.
Methods
Materials
Human vascular SMCs derived from aorta were purchased from the
American Type Culture Collection. Kaighn’s modification of Ham’s
F12 (F12K) medium, FCS, trypsin-EDTA, insulin, transferrin, and
selenium were purchased from Invitrogen. Antibodies to cyclins A,
B, and D3, and CDKs 1 and 2 were from BD Biosciences. 2-ME,
anti-p27, anti-phospho-histone H3 (ser10), and anti-␣-actin were
from Sigma-Aldrich.
Cell Culture
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Human vascular SMCs were cultured in Kaighn’s modification of
Ham’s F12 (F12K) medium containing 10% FCS and supplemented
with insulin–selenium–transferrin. The identity of SMCs was confirmed by immunostaining with antibody against smooth muscle
␣-actin. After seeding in the F12K medium with 10% serum for 48
hours, cells were treated with or without 2-ME for time periods
indicated in the relevant figures. For cell proliferation experiments,
the culture medium was changed every 2 days for up to 6 days; cell
numbers were counted with a hemocytometer.
Cell Cycle Analysis by LSC
Cell cycle analysis and 5-bromodeoxyuridine (BrdUrd) incorporation were performed using laser scanning cytometer (CompuCyte
Corp), as described previously.12 In addition, BrdUrd pulse-chase
assay was performed to track the position changes of the BrdUrdpositive cells in cell cycle phases.
Immunofluorescence Studies
For most immunocytochemical experiments, cells grown on
coverslips were fixed with 4% paraformaldehyde for 20 minutes
at room temperature and permeabilized with 100% methanol
overnight at ⫺20°C. Cells were then washed with PBS and
blocked with 2% skim milk for 30 minutes. For studies of mitotic
index, cells were stained with an antibody (1:300 dilution) against
phosphohistone H3 (serine 10), a mitotic marker. Secondary
antibody (1:400 dilution) was anti-rabbit IgG conjugated to
Texas-Red. The nuclei were counterstained with 4⬘, 6-diamidino2-phenylindole (DAPI; 10 mg/mL in PBS) for 20 minutes. Cells
were inspected by fluorescence microscopy and photographed
with a CCD camera. Mitotic cells were identified by the presence
of condensed DNA and by phosphohistone H3 (ser 10)–positive
staining. The mitotic index was calculated as the percentage of
mitotic cells versus the total cell count.
For experiments regarding the microtubule network, mitotic
spindle, and centrosome, anti-␣-tubulin and anti-␥-tubulin were
used as primary antibodies and anti-mouse IgG-Alexa Fluor 488
and anti-rabbit IgG-Texas Red as secondary antibodies, respectively. Confocal images were acquired with a laser scanning
confocal microscope (Leica Microsystems) under a ⫻100 oil
immersion lens and photographed with Cooled Scientific CCD
camera (Princeton Instruments). Stacked images collected at
0.8-mm planes were analyzed and processed for deconvolution by
Imaris software.
Assays for Apoptosis
DNA fragmentation analysis of SMCs treated with or without 2-ME
at the concentrations indicated for 48 hours was performed as we
described previously.13 Live cell apoptosis was performed using
Annexin V-FITC apoptosis detection kit (Sigma) according to the
manufacturer’s recommendations. To determine whether mitotic cells
are apoptotic, cells were subjected to TUNEL assay and phosphohistone H3 staining. The TUNEL assay was modified from the APOBrdUrd TUNEL assay kit (Molecular Probes).
Western Blot Detection
SMCs grown in 100-mm Petri dishes after treatment as desired, were
lysed, and equal amounts of protein from each sample were separated by 11% SDS-PAGE as described previously.12 Antibodies
against p27, CDKs 1 and 2, and cyclins D3, A, B, and E were used
to detect the respective proteins.
Statistical Analysis
Results represent the mean value (mean⫾SEM) of experiments as
indicated in the relevant figures. Statistical comparisons were performed
with the Student t test for unpaired observations or with ANOVA
followed by Bonferroni’s correction for comparisons of ⱖ3 groups. P
value ⬍0.05/n (where n is the number of comparisons) was considered
to indicate a statistically significant difference.
Results
2-ME Inhibits Proliferation and Induces
Apoptosis of Human Vascular SMCs
To determine the effects of 2-ME on SMC growth, human
SMCs were cultured for ⱕ6 days in 10% serum in the
absence and presence of 2-ME (0.1, 1, and 10 ␮mol/L). We
Figure 1. 2-ME inhibits proliferation and
induces apoptosis of human vascular
SMCs. Human SMCs were seeded at a
density of 10 000 cells/well in 12-well
plates and cultured in F12K medium
containing 10% FBS for 24 hours. After
changing to fresh medium (day 0), cells
were treated without or with different
concentrations of 2-ME for up to 6 days.
During this period, the culture medium
was changed at 48-hour intervals. Viable
cells were counted every day using a
hemocytometer. Values are expressed
as mean⫾SEM (n⫽5; A). *Pⱕ0.05 and
**Pⱕ0.001. Cells, after treatment without
or with 2-ME for 48 hours, were lysed
for DNA extraction. DNA (10 ␮g/lane)
was loaded onto 1.8% agarose gels (B).
M indicates 100-bp DNA ladder marker.
Gui and Zheng
found that viable cell numbers were reduced in response to
0.1 ␮mol/L 2-ME compared with the control cells without the
treatment of 2-ME (P⬍0.05). Viable cell numbers were even
more significantly decreased in the presence of 1 ␮mol/L and
10 ␮mol/L 2-ME compared with the control cells (P⬍0.001;
n⫽5; Figure 1A). After 6 days of treatment with 10 ␮mol/L
2-ME, the viable cells decreased to 0.5⫾0.2⫻104 versus the
seeding level of 1.0⫻104 (P⬍0.01; n⫽5), suggesting a
cytotoxic effect.
To investigate whether the cytotoxic effect induced by
2-ME involves induction of apoptosis, DNA fragmentation, a
hallmark of apoptosis, was assayed. Human SMCs treated with
2-Methoxyestradiol and Human Smooth Muscle Cells
3
1 ␮mol/L 2-ME displayed a typical DNA laddering pattern,
which was enhanced in the cells treated with 10 ␮mol/L 2-ME
(Figure 1B). These results indicate that 2-ME causes apoptosis
in human SMCs.
2-ME Inhibits DNA Synthesis and Induces
Accumulation of Cells With 4N DNA Content
To explore whether the antiproliferative effect of 2-ME on
human SMCs is through a mechanism regulating the cell
cycle, cell cycle profiles in response to 2-ME were examined
by LSC. Human SMCs were stimulated with 2-ME (0.1, 1,
and 10 ␮mol/L) for up to 48 hours. Cells in S phase, which
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Figure 2. Effects of 2-ME on BrdUrd
incorporation and cell cycle profile. After
treatment with 2-ME (0.1, 1, and
10 ␮mol/L) for 24 hours, cells were
labeled with 10 ␮mol/L BrdUrd for
1 hour and then fixed for detection with
anti-BrdUrd antibody and PI staining
using LSC. Representative scattergrams
show BrdUrd staining intensity (y axis,
FITC integral) vs DNA content (x axis, PI
integral). BrdUrd-positive cells are distributed in the regions of upper quadrants (A). Histograms represent the number of cells vs DNA content (B). 2N and
4N indicate cells with 2N and 4N DNA
contents.
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Effects of 2-ME on Cell Cycle Profile
6 Hours
12 Hours
24 Hours
48 Hours
Groups
2N
S
4N
2N
S
4N
2N
S
4N
2N
S
4N
Control
68⫾4
28⫾3
5⫾3
68⫾4
27⫾4
5⫾2
69⫾5
25⫾4
6⫾3
72⫾4
23⫾4
5⫾2
0.1 ␮mol/L
66⫾5
27⫾4
7⫾3
67⫾4
26⫾2
6⫾2
70⫾3
23⫾3
6⫾2
83⫾4
16⫾3
5⫾2
1 ␮mol/L
60⫾3
25⫾3
15⫾2
64⫾4
21⫾3
15⫾2
75⫾4
16⫾3
8⫾3
84⫾5
8⫾3
8⫾3
10 ␮mol/L
57⫾5
21⫾3
20⫾4
50⫾5
7⫾3
35⫾4
41⫾4
13⫾3
47⫾4
36⫾4
6⫾4
60⫾5
Summarized data (n⫽5) of cells with 2N and 4N DNA contents (2N and 4N cells) and cells in S phase (the BrdUrd-positive cells) in response to different time
treatments with 2-ME (0.1 to 10 ␮mol/L). Numbers indicate percentage values⫾SEM.
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are synthesizing DNA actively, were detected by incorporation of BrdUrd. The scattergram shows that BrdUrd-positive
cells (high FITC integral) were reduced from 12 to 48 hours
in the presence of 1 and 10 ␮mol/L 2-ME (Figure 2A and
Table). Cell distributions in cell cycle phases according to
their DNA contents are displayed as histograms in Figure 2B.
The histogram shows that treatment with 0.1 ␮mol/L 2-ME
did not cause a significant change in the cell cycle distribution compared with the control. In contrast, treatment with
10 ␮mol/L 2-ME resulted in accumulation of cells with ⱖ4N
DNA content (4N cells) as early as 6 to 12 hours, which was
more significant at 24 to 48 hours (Figure 2B and Table 1).
The increase in 4N cells coincided with a decrease in 2N
cells. However, treatment with 1 ␮mol/L 2-ME only caused
a temporary (at 6 to 12 hours) increase in cells with ⱖ4N
DNA content. Cell cycle profiles in response to 2-ME are
summarized in the Table. Note that the decrease in the S
phase (BrdUrd-positive) cells in response to 2-ME treatment was concentration and time-dependent. The accumulation of cells with ⱖ4N DNA content in the presence of
10 ␮mol/L 2-ME was also time-dependent. These results
suggest that 2-ME arrests cells at cell cycle stages with
ⱖ4N DNA content.
after 24 hours and 48 hours of treatment indicates that a
fraction of the mitotic cells might have undergone mitotic
slippage.
2-ME Induces Mitotic Slippage and Endoreduplication,
Which Is Associated With an Increase in CDK2 and a
Reduction in p27 Expression
To evaluate whether accumulation of 4N cells was because of
mitotic slippage, BrdUrd pulse-chase assays were carried out
to trace changes of the cell cycle position of BrdUrd-positive
cells after S phase as described in the Methods section. As
shown by the representative results (Figure 4A), in the control
groups, the majority of BrdUrd-positive cells started to arrive
at the 2N region after 6 hours and significantly accumulated
at the 2N region at 12 hours, indicating that those cells had
already finished cell division and become daughter cells in
G1/0 phases. At 24 hours, some of the daughter cells had
entered into the G2/M phase of the next cell cycle as they
appeared at the 4N region. In contrast, in the groups treated
2-ME Induces Cell Cycle Arrest
in the Mitotic Phase
Cells with ⱖ4N DNA content could be in the G2 phase,
mitotic phase, or postmitotic phases (tetraploidy). To evaluate
whether 2-ME induces cell cycle arrest at mitosis, we stained
cells with phosphohistone H3 (serine 10), a mitotic marker,
and DAPI, a DNA-specific dye. Mitotic cells, containing
condensed DNA and positive for phosphohistone H3, were
significantly increased in the presence of 1 to 10 ␮mol/L
2-ME up to 24 hours (Figure 3). In response to 2-ME
treatment (1 to 10 ␮mol/L), mitotic cells began to increase at
3 hours of treatment, reached a maximum at 6 hours, and then
slowly declined but remained above the untreated control
(Figure 3). However, treatment with 0.1 ␮mol/L 2-ME only
temporarily (at 3 to 6 hours) induced a small increase in
mitotic cells (Table). Summarized results showed that in the
presence of 10 ␮mol/L 2-ME, the mitotic index was 22⫾3%
after 6 hours of treatment, then decreased to 12⫾2% and
8⫾2% at 24 hours and 48 hours, respectively. Note that in the
presence of 10 ␮mol/L 2-ME for 24 hours and 48 hours, cells
with ⱖ4N DNA content were 47⫾4% and 60⫾5% (Table);
therefore, not all 4N cells are mitotic cells. The decline in
mitotic index coincident with the accumulation of 4N cells
Figure 3. 2-ME induces mitotic arrest in human SMCs. Human
SMCs, treated without or with 2-ME at the concentrations for
indicated time periods, were fixed with 4% paraformaldehyde
and stained for ␣-tubulin, phosphohistone H3, and DAPI. Mitotic
cells were observed containing condensed DNA by DAPI staining, which were in agreement with positive staining for phosphohistone H3. The mitotic index in response to 2-ME treatment
for ⱕ48 hours was measured as the percentage of cells with
condensed DNA and positive for phosphohistone H3 staining vs
total cell count. At least 500 cells were scored from each coverslip. Values are mean⫾SEM (*Pⱕ0.001; n⫽4).
Gui and Zheng
2-Methoxyestradiol and Human Smooth Muscle Cells
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Figure 4. 2-ME induces mitotic slippage and endoreduplication in human SMCs. Cells were first labeled with 10 ␮mol/L BrdUrd for
1 hour and then cultured in BrdUrd-free growth medium for up to 24 hours followed by fixation at different time points and detection of
BrdUrd-positive cells (A). Cells distributing in the upper quadrants (high FITC value) represent BrdUrd-positive cells. Cells were divided
in 2N and 4N regions according to their DNA contents. Inserts were photographed from cells on the same coverslips as were scanned
by LSC. BrdUrd-positive cells are shown in green; DNA is shown in red. Cells, after treatment with 2-ME at different concentrations for
48 hours, were labeled with BrdUrd for 1 hour, and fixed for detection of the incorporated BrdUrd (in green). Nuclei were counterstained with PI (red). Note that some of the polyploid nuclei appear as BrdUrd-positive (arrows) in the cells treated with 1 ␮mol/L and
10 ␮mol/L 2-ME (B), indicating the occurrence of endoreduplication. Cells, treated with and without 2-ME, were lysed for protein
extraction. Samples (75 ␮g protein/lane) were subjected to 11% SDS-PAGE and Western blotting for detection of specific proteins as
indicated. Representative results from 4 independent experiments are shown (C). ␣-Actin was used as a loading control.
with 1 and 10 ␮mol/L 2-ME, most of the BrdUrd-positive
cells had arrived at the 4N region by 6 hours, and stayed there
until 24 hours. After 24 hours of treatments, the majority of
BrdUrd-positive cells left for the 2N region in the group
treated with 1 ␮mol/L 2-ME but not in the group treated with
10 ␮mol/L 2-ME. Corresponding images to the scanned cells
in each group after 24 hours of treatment (Figure 4A, inserts)
show that some of the BrdUrd-positive cells exhibited
polyploidy or micronuclei in the presence of 1 and 10 ␮mol/L
2-ME. These results suggest that, in response to 2-ME, cells
underwent mitotic slippage after the exit from mitotic arrest.
In order to determine whether mitotic cells pursue endoreduplication, we next searched for aneuploidy cells that
continually synthesized DNA in response to 2-ME. In the
cells treated with 1 to 10 ␮mol/L 2-ME for 24 hours, BrdUrd
was added for 1 hour to label S-phase cells. We observed that
some of the aneuploid cells were BrdUrd positive (Figure 4B,
arrows), indicating that 2-ME treatment induces endoreduplication in human SMCs. Because not every 4N cell could be
distinguished by microscopy, we were unable to quantify how
many 4N cells were in the S phase. In searching for other
supportive evidence, we examined the expressions of the cell
cycle regulatory proteins cyclins D3, B, A, and E; CDKs 1
and 2; and the CDK inhibitor p27. Representative results
from 4 sets of independent experiments showed that CDK2
was increased significantly in response to 1 and 10 ␮mol/L
2-ME. The increase in CDK2 protein level was concurrent
with a decrease in p27 expression (Figure 4C). The expression levels of CDK1 and cyclins A, B, E, and D3 were not
changed by the presence of 2-ME.
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Figure 5. Concentration-dependent effects of 2-ME on the microtubule network, mitotic spindle, and centrosome integrity. Human
SMCs were treated with 2-ME at the concentrations indicated for 24 hours, fixed with 100% methanol, and then stained with anti-␣tubulin and PI (A). The microtubule network was examined by fluorescence microscopy. After exposure to 2-ME for 1 hour, cells were
fixed and stained with anti-␣-tubulin, anti-␥-tubulin, and DAPI. The images for the mitotic spindle, centrosome, and chromosome of
cells in mitosis and cytokinesis were acquired by confocal microscopy (B). Cells in interphase depicted (C) were examined after treatment with 2-ME for 12 hours. Microtubule network and mitotic spindles are in green; centrosomes are red; chromosomes are blue.
Gui and Zheng
2-Methoxyestradiol and Human Smooth Muscle Cells
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Figure 6. Mitotic cells undergo apoptosis in response to 2-ME. Live human SMCs, incubated with different concentrations of 2-ME for
6 hours, were stained with Annexin V, PI, and Hoechst for 10 minutes and inspected by fluorescence microscopy. For the bottom 3
panels, cells were pretreated with thymidine for 12 hours before addition of 2-ME. Images represent 3 sets of independent experiments
(A). White arrows indicate Annexin V positive/PI negative (early apoptotic cells). Cells, treated with and without 2-ME for 6 hours, were
fixed with 4% paraformaldehyde and detected with antiphosphohistone H3 and TUNEL assay (B). DNA was counterstained with DAPI.
Note the excellent agreement of the signals for antiphosphohistone H3 with TUNEL staining. Percentages of mitotic cells and corresponding TUNEL-positive cells were displayed (C). For each coverslip, ⱖ500 cells were counted. **Increases in mitotic index are significant when compared with the control (Pⱕ0.001; n⫽4). *Differences in TUNEL-positive cells between the 2-ME–treated groups and control were significant (Pⱕ0.001, n⫽4). #, ##Pⱕ0.001 when comparing groups treated with 10 ␮mol/L 2-ME with groups pretreated with
thymidine (n⫽4).
2-ME Induces Damage of Mitotic Spindle, Impact
of Centrosome and Chromosome Integrity,
and Aberrant of Cytokinesis
To explore the cellular mechanism underlying the mitotic
arrest induced by 2-ME, given that 2-ME can inhibit tubulin
polymerization and disrupt microtubule function,11 we examined the effects of 2-ME on the microtubule network, mitotic
spindle, and centrosome integrity by detection of immunofluorescent signals of ␣-tubulin and ␥-tubulin, a centrosomal
component. Representative results showing the effects of
2-ME on the omicrotubule network in response to 2-ME are
illustrated in Figure 5A. The microtubule network remained
intact in the presence of 0.1 and 1 ␮mol/L 2-ME. However,
after treatment with 10 ␮mol/L 2-ME, the microtubule network
exhibited a disorganized structure, distributing at the membrane region and perinuclear cytoplasmic regions. The gap
between those 2 regions indicates tubulin disrupted by 2-ME.
In addition, note that mitotic microtubule bundles in the
presence of 0.1 ␮mol/L 2-ME displayed bipolar spindle
morphology. In contrast, in the presence of 1 ␮mol/L 2-ME,
the mitotic microtubule appeared as irregular multipolar
bundles. The effects of 2-ME on the mitotic spindle at the
early stage were additionally examined by confocal microscopy. Representative confocal images of cells incubated in
the absence and presence of 2-ME for 30 minutes to 1 hour
are shown in Figure 5B. Control mitotic cells have normal
bipolar spindles, 2 pairs of centrosomes each of which attach
to a spindle pole, and chromosomes aligned to the metaphase
plate. Treatment with 0.1 ␮mol/L 2-ME did not cause an
obvious change in mitotic cells. However, after treatment
with 1 ␮mol/L 2-ME, mitotic cells displayed irregular multipolar spindles, metaphase plate, and disoriented chromosome, concurrent with the appearance of multipolar centrosomes, which are still attached to the poles. In cells treated
with 10 ␮mol/L 2-ME, the mitotic spindle and metaphase
plate were disrupted completely; centrosomes appeared in the
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middle of the chromosomes, which seemed improperly
packed. In the cytokinesis process, the control cells divided
into 2 equal daughter cells, each of which contains a centrosome, a chromosome, and a bundle of mitotic spindles connecting to the midbody. In the presence of 0.1 ␮mol/L 2-ME,
cells were still able to divide into 2 equal daughter cells, but
1 of them has 2 centrosomes, whereas the other has only 1. In
response to 1 ␮mol/L 2-ME, cells divided multipolarly with
multiple spindles, overamplified centrosomes, and improperly segregated chromosomes. Damaged spindle bundles
were linked to the midbody. One daughter cell has 2 chromosomes and 3 centrosomes, whereas the other has 3 centrosomes without a chromosome. In the cells treated with
10 ␮mol/L 2-ME, spindles were completely disrupted, chromosomes asymmetrilly segregated or failed to segregate, and
the midbody was missing. In this case, if cytokinesis occurred, 1 daughter cell would have double the number of
chromosomes, whereas the other would have none. Indeed,
we observed the appearance of cleaved cells with a centrosome
but without a chromosome (data not shown). Furthermore, after
treatment with 2-ME for 1 hour, the interphase cells did not
exhibit abnormal microtubule spindle, centrosomal, and chromosomal structure (data not shown). Studies were carried out to
inspect the interphase cells in the groups exposed to 2-ME for 12
hours, when 4N cells were accumulated and mitotic cells
declined. As shown in Figure 5B (right), the interphase cells in
the 0.1 ␮mol/L 2-ME–treated group seemed to have normal
microtubules, centrosomes, and chromosomes. In contrast, in the
1 to 10 ␮mol/L 2-ME–treated groups, the interphase cells were
multinuclear, containing multiple centrosomes.
Mitotic Arrest Cells in Response to the
Treatment of 2-ME Undergo Apoptosis
Based on the finding that human SMCs displayed a DNA
laddering pattern in response to 2-ME treatment (Figure 1B)
to further characterize the apoptotic effect induced by 2-ME,
we evaluated apoptosis in live cells (Figure 6A) with Annexin
V and propidium iodide (PI) staining. To visualize all of the
cells, Hoechst dye, a cell-permeable DNA dye, was also
applied during the staining. The results show that in the
presence of 2-ME (1 and 10 ␮mol/L) for 6 hours, cells that
had condensed DNA as detected by Hoechst dye were
Annexin V-positive and/or PI positive, suggesting that these
mitotic cells were apoptotic. In addition, to see whether
blockage of entry into mitosis would affect apoptosis, we
pretreated cells with thymidine for 12 hours to synchronize
cells at the boundary of G1 to S phase14 and then added
10 ␮mol/L 2-ME in the presence of thymidine for another 6
hours. Preincubation of thymidine for 12 hours, which arrested
93% of cells in the G1/0 phase (data not shown), significantly
reduced the Annexin V– and PI–positive cells induced by
10 ␮mol/L 2-ME (Figure 6A). Interestingly, the cells with
multiple nuclei were Annexin V and PI negative when treated
with 2-ME for 6 hours (data not shown), implying that the
aneuploid cells are nonapoptotic at this stage. Next, in order to
confirm that the mitotic cells were apoptotic, we stained the cells
with both phosphohistone H3 (a mitosis marker) and TUNEL
(an apoptosis marker). The nuclei were counterstained with
DAPI. The increased mitotic cells identified by both DAPI and
phosphohistone H3 stainings were in excellent agreement with
those positive for TUNEL staining in the presence of 2-ME
(Figure 6B). Our accumulated data showed that 0.1 ␮mol/L
2-ME increased TUNEL-positive cells, although there was no
significant DNA fragmentation detected (Figure 1B). The
TUNEL-positive cells after treatment of 10 ␮mol/L 2-ME were
significantly reduced in the group synchronized in G1/0 phases by
thymidine (Figure 6C). In addition, the presence of thymidine
prevented the reduction of p27 and increase of CDK2 in
response to 2-ME treatment (data not shown). Quantitative data
of the percentage of TUNEL-positive cells with concurrent
mitotic index in response to 6 hours of 2-ME treatment at
different concentrations are shown in Figure 6C. Note that in
response to 2-ME treatment, the increase in mitotic cells coincided with the induction of apoptosis. The presence of thymidine
completely inhibited apoptosis induced by 2-ME.
Discussion
Antiproliferative effects of 2-ME have been found in a
variety of cell types, including human breast cancer, myeloma, and endothelial cells.9 The process by which 2-ME
affects cell growth may involve arresting cells in G2/M and
inducing apoptosis. In human aortic SMCs, it has been shown
that 2-ME mediated the inhibitory effects of estradiol on cell
proliferation and DNA synthesis independent of estrogen
receptor.10 It has also been reported in an abstract that 2-ME
induced both G2/M and G1/0 arrest based on flow cytometry
data.15 However, the precise cell cycle phase at which 2-ME
arrests human SMCs has not been documented. Our data
demonstrate the sequential events that occur in human SMCs
in response to 2-ME. In the presence of 2-ME (1 to
10 ␮mol/L) for as little as 30 minutes to 1 hour, the mitotic
spindle was damaged, and centrosomal and chromosomal
integrity were affected. Exposure of cells to 2-ME for 1 to 3
hours led to aberrant cytokinesis. Subsequently, increases in
mitotic cells were detected after 3 to 6 hours of treatment,
which coincided with apoptosis in mitotic cells detected with
the TUNEL assay. Additionally, mitotic cell apoptosis induced by 2-ME was prevented by synchronization of cells in
G1/0 with thymidine. These results suggest that the antiproliferative effect of 2-ME is through induction of cell cycle arrest
in mitosis and promotion of mitotic cell apoptosis, and the
cellular mechanisms underlying 2-ME–induced effects involve rapid disruption of the mitotic spindle and centrosomal
integrity. Along with evidence that 2-ME can interact with
tubulin directly to inhibit tubulin polymerization,11 it is
possible that 2-ME activates the spindle checkpoint and
induces the onset of apoptosis in mitotic cells. However, how
activation of the spindle checkpoint promotes apoptosis and
whether phosphorylation of histone H3 (ser 10) during mitosis
plays a role in mitotic cell apoptosis are unknown, although
phosphorylation of histone H2B at serine 14 by mammalian MstI
kinase has been linked to mitotic cell apoptosis.16 Given that
aurora kinases can phosphorylate histone H3 at both serine 10
and serine 28, it will be interesting to see whether aurora kinases
play a role in mitotic cell apoptosis induced by 2-ME.
It has been shown that most mammalian cells undergo
adaptation of the mitotic spindle checkpoint in the presence
of microtubule-disrupting agents.17 Once cells adapt to the
Gui and Zheng
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spindle checkpoint, they can aberrantly exit from mitosis and
enter interphase in a tetraploidy state. In our studies, the
induction of mitotic arrest and aberrant cytokinesis by 2-ME
may contribute to the accumulation of 4N cells observed after
6 to 12 hours of treatment. The significant accumulation of
4N cells after 24 to 48 hours of treatment with 10 ␮mol/L
2-ME may result from the processes of mitotic slippage and
endoreduplication. This is supported by the following findings: (1) declined mitotic index occurs concurrent with the
accumulation of 4N cells; (2) BrdUrd-positive cells delayed
entry into the next cell cycle and turned into aneuploid or
micronuclear cells; and (3) some aneuploid cells continued to
synthesize DNA. In addition, the increase in CDK2 expression and the reduction of p27 in response to 2-ME may also
support the occurrence of endoreduplication. This observation is consistent with the finding that loss of CDK inhibitors
p21 and p27 results in upregulation of CDK2 activity during
endoreduplication induced by mitotic spindle disruption.7
Another possibility is that CDK2 plays a role in apoptosis
independent of its roles in the normal cell cycle, as observed
in cardiomyocyte apoptosis.18 Furthermore, our studies suggest that polyploidy or multiple nuclei could rise from more
than two different mechanisms: multipolar cleavages with
uneven amounts of chromosomes as seen in cells treated with
1 ␮mol/L 2-ME or failure of chromosome segregation because of complete disruption of the mitotic spindle as seen in
cells treated with 10 ␮mol/L 2-ME. Therefore, after mitotic
slippage, 4N cells induced by 1 ␮mol/L 2-ME only temporally increased. Because of uneven chromosomal segregation,
the daughter cells could have ⬍4N DNA content. In contrast,
the chromosome was not segregated in response to
10 ␮mol/L 2-ME, and 4N cells could propagate as tetraploid/
polyploidy cells.
In response to 2-ME, mitotic slippage and endoreduplications leading to the formation of polyploidy and aneuploidy
could potentially increase the genomic instability of vascular
SMCs. Aneuploidy has been documented to be a characteristic of a variety of human tumors,17 and genetic instability of
aneuploid cells has been proposed to contribute to the
development of cancer.19 In addition, polyplidization has
been reported to be associated with hypertrophic vascular
SMCs in hypertensive animals and patients.20,21 An increase
in apoptosis of vascular SMCs is also documented in the
arterial wall of hypertension.22 Therefore, it will be important
to determine whether 2-ME effects on vascular SMCs could
contribute to the pathogenesis of vascular hypertensive disease.
The effective concentration of 2-ME for inhibiting human
SMC proliferation and inducing apoptosis in the present
study was between 0.1 and 1 ␮mol/L, which is similar to the
concentration used to inhibit endothelial cell growth and
angiogenesis in vitro by others.23 These effective concentrations of 2-ME seem much higher than that in normal human
plasma,8 which is in the range of picomolars to tens of
nanomolars. However, it has been found that vascular SMCs
from one origin can more effectively metabolize estradiol to
generate 2-ME than those from another.24 Note that 2-ME has
been used at the concentrations ⬎1000 mg/day in participants
in clinical trials.25 It is possible, therefore, that lipophilic
2-ME might reach higher concentrations in some cellular
2-Methoxyestradiol and Human Smooth Muscle Cells
9
regions than in the plasma. In addition, the finding that the
rapid effect of 2-ME is to induce mitotic cell arrest and
apoptosis through impact on the mitotic spindle, whereas
SMCs usually remain in a quiescent cell cycle stage in intact
vessels, raises an important question of whether 2-ME affects
vascular SMCs in vivo. As we know, under certain pathophysiological conditions, such as atherosclerosis and restenosis, vascular SMCs are highly proliferative. Inhibition of cell
cycle progression has been found to prevent the development
of these diseases.1 Therefore, it will be important to examine
the effects of 2-ME in models of postangioplasty restenosis
and atherosclerosis in vivo. Under in vivo condition, the
concentration of 2-ME could be much lower, and it is also
possible that the mechanisms of antiproliferative actions of
2-ME, if any, may be through other mechanisms independent
of apoptosis. Our in vitro study indicates that 0.1 ␮mol/L
2-ME could induce an abnormal centrosome number in cells
after cytokinesis (Figure 5). As documented, aberrant centrosome itself can cause genomic instability and tumor progression.26 However, we cannot determine whether the lower
concentrations of 2-ME have nonantiproliferative actions,
because it has been reported recently that 2-ME stimulated
the growth of breast cancer cells through activation of the
estrogen receptor.27 Taken together, our data have demonstrated the antiproliferative effect of 2-ME on human vascular
SMCs in vitro, which certainly allows us to better understand
the pathological roles of 2-ME.
Acknowledgments
This work was supported by a grant from the Heart and Stroke
Foundation of Alberta (to X.-L. Z.). X.-L. Z. and Y.G. are recipients
of a New Investigator Award and a Postdoctoral Fellowship Award,
respectively, from the Heart and Stroke Foundation of Canada. We
are very grateful to Dr Michael P. Walsh for advice during the course
of this work and in preparation of the article and for providing the
LSC through his Tier I Canada Research Chair and associated
equipment grant from the Canada Foundation for Innovation with
matching funds from Alberta Innovation and Science Research
Infrastructure, the Alberta Heritage Foundation for Medical Research, and the University of Calgary.
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2-Methoxyestradiol Induces Cell Cycle Arrest and Mitotic Cell Apoptosis in Human
Vascular Smooth Muscle Cells
Yu Gui and Xi-Long Zheng
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Hypertension. published online December 27, 2005;
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